Retinoid induced gene

ABSTRACT

A novel human cDNA, called TIG3 (Tazarotene Induced Gene 3), inducible by RAR-selective retinoids and structurally related to a known tumor suppressor gene. Methods of detecting the TIG3 polynucleotide.

This application is a continuation of U.S. patent application Ser. No.08/808,303, filed Feb. 28, 1997 now U.S. Pat. No. 5,776,687.

FIELD OF THE INVENTION

The present invention relates generally to the field of inducible geneexpression. More specifically, the invention relates to aretinoid-inducible polynucleotide and assays that detect expression ofthis polynucleotide.

BACKGROUND OF THE INVENTION

Retinoids, which are the compounds comprising vitamin A and itsderivatives, play important roles in a variety of biological phenomena.More particularly, retinoids are important for vision, hematopoiesis,bone development and pattern formation during embryogenesis. Retinoidsalso exhibit antiproliferative activities in certain biologicalcontexts.

Retinoids also have been used extensively as pharmaceutical agents fortreating various malignant and non-malignant skin diseases. Malignantskin diseases therapeutically responsive to retinoids include squamouscell carcinoma, actinic keratoses, basal cell carcinoma and Kaposi'ssarcoma. Additionally, retinoids are potentially useful aspharmacological agents for the treatment of various epithelial cancers(Peck and DiGiovanna, “Synthetic Retinoids in Dermatology”) in TheRetinoids, 2nd ed., pp 631-658 (1994); Boehm et al., Exp. Opin. Invest.Drugs 4:593 (1995); Nagpal and Chandraratna, Curr. Pharm. Design 2:295(1996)). Examples of non-malignant skin diseases therapeuticallyresponsive to retinoids include psoriasis and acne. In spite of thedemonstrated utility of this class of pharmacological agents, themolecular basis of retinoid action in skin and various cancers is poorlyunderstood.

Two families of nuclear receptors, called the retinoic acid (RA)receptors (RAR-α, -β and -γ) and the retinoid X receptors (RXR-α, -β and-γ), mediate pharmacological and physiological retinoid signalling(Chambon, Sem. in Cell Biol. 5:115 (1994); Mangelsdorf et al., “TheRetinoid Receptors” in The Retinoids, 2nd ed., pp 319-349 (1994); Boehmet al., Exp. Opin. Invest. Drugs 4:593 (1995); Nagpal and Chandraratna,Curr. Pharm. Design 2:295 (1996)). RARs and RXRs, which belong to thesuperfamily of steroid/thyroid/vitamin D₃ nuclear receptors, readilyheterodimerize in vitro (for references see, Nagpal and Chandraratna,Curr. Pharm. Design 2:295 (1996)) and function as heterodimers in vivo(Nagpal et al., EMBO J 12:2349 (1993)). These receptors areligand-dependent transcription factors which activate the expression ofretinoid responsive genes by cooperative action of their activationfunctions. These activation functions are called AF-1, aligand-independent activation function, and AF-2, a ligand-dependentactivation function (Nagpal et al., EMBO J. 12:2349 (1993)).

The two families of retinoid receptors differ from each other withrespect to the ligands that bind and activate the receptors.All-trans-RA (RA) binds and activates the RAR family of receptors. Adifferent ligand, 9-cis-RA (9C-RA), binds and activates both the RARsand members of the retinoid X receptor (RXR) family. The retinoid calledAGN 190168 (Tazarotene/ethyl 6-[2-(4,4) dimethyl-thiochroman-6-yl]ethynyl-nicotinate) is one example of an RAR-β/γ selective syntheticretinoid having therapeutic utility. More specifically, this syntheticretinoid can be administered topically to dramatically improve thesymptoms associated with psoriasis.

Only a small number of retinoid-inducible gene products have beenidentified to date. Of these, the gene product encoding a cellularretinoic acid binding protein, called CRABP II, is the only marker knownto be induced in vivo by RA in non-diseased skin (Elder et al., JInvest. Dermatol. 100:356 (1993)). Interestingly, CRABP II expressionwas down-regulated by RA in submerged keratinocyte cultures (Elder andCromie, J. Toxicol.—Cut. & Ocular Toxicol. 12:173 (1993)) and wasoverexpressed in cells of tissues that exhibited a psoriatic phenotype(Didierjean et al., Biochem. Biophys. Res. Comm. 180:204 (1991)). Thosehaving ordinary skill in the art will appreciate that psoriasis is ahyperproliferative and inflammatory condition of the skin (Krueger andDuvic, J Invest. Dermatol. 102:14S (1994)) which clinically responds toretinoid treatment (Esgleyes-Ribot et al., J Am. Acad. Dermatol. 30:581(1994); Weinstein, Brit. J Dermatol. 135(Suppl. 49):32 (1996)).

Two novel genes, called Tazarotene-induced gene 1 (TIG1) andTazarotene-induced gene 2 (TIG2), were recently identified by virtue oftheir inducible expression in skin raft cultures treated with Tazarotene(Nagpal et al., J Invest. Dermatol. 106:269 (1996); Nagpal et al.,submitted (1997)). TIG1 was also shown to be induced by Tazarotene inforeskin keratinocyte and fibroblast cultures. Significantly, both TIG1and TIG2 were induced in vivo by topical treatment of psoriatic lesionswith Tazarotene.

Herein we disclose the discovery and utility of a novel retinoid-inducedpolynucleotide that is unrelated to either TIG1 or TIG2.

SUMMARY OF THE INVENTION

One aspect of the present invention regards an isolated polynucleotidethat encodes a protein having the polypeptide sequence of SEQ ID NO:12.In a preferred embodiment the polynucleotide has the sequence of SEQ IDNO:11.

A second aspect of the invention regards a method of identifying a testcompound for treatment of a hyperproliferative disorder of skin.According to the invented method, a negative control sample containingRNA isolated from an untreated control culture of cells derived fromskin is first obtained. The cells of this control culture have not beentreated with an inducer. Next, a test sample containing RNA isolatedfrom a test culture of said cells derived from skin is obtained. Cellsin this test culture will have been treated with the test compound.After the two samples of RNA have been obtained, the amount ofTazarotene Inducible Gene-3 (TIG3) RNA present in each of the samples isquantitated. The TIG3 RNA is an RNA having a polynucleotide sequencecorresponding to the sequence of SEQ ID NO:11. Finally, the amount ofTIG3 RNA in each of the samples is compared to determine if the amountof TIG3 RNA in the test sample is greater or lesser than the amount ofTIG3 RNA in the negative control sample. A compound will be identifiedas a test compound for the treatment of the hyperproliferative disorderif the amount of TIG3 RNA in the test sample is greater than four-foldmore than the amount of TIG3 RNA in the negative control sample. In apreferred embodiment, the step for quantitating TIG3 mRNA will involvehybridizing the negative control sample and the test sample with alabeled probe having a sufficient number of consecutive nucleotidescomplementary to the sequence of SEQ ID NO:11 to specifically hybridizewith TIG3 mRNA under high stringency conditions (0.1×SSPE/1% SDS at 65°C.), and then quantitating the amount of hybridization between the probeand each of the samples. In another preferred embodiment, the negativecontrol sample is derived from keratinocytes or fibroblasts. In yetother preferred embodiments, the TIG3 probe used in the procedures islabeled with a radioactive label and the amount of hybridized probe isquantitated by autoradiography. RNA contained in the negative controlsample and RNA contained in the test sample can be immobilized to asolid support prior to the hybridizing step. In a different embodimentof the invented method, the step for quantitating the amount of TIG3 RNApresent in negative control and test samples is accomplished by: (i)reverse transcribing mRNA present in each of the samples, wherein theproduct of reverse transcription has a polynucleotide sequencecorresponding to a segment of the sequence given by SEQ ID NO:11, theproduct being TIG3 cDNA; (ii) amplifying specifically any TIG3 cDNAproduced in step (i) by a polymerase chain reaction to result in theproduction of TIG3 amplification products; and (iii) quantitating theTIG3 amplification products produced when negative control and testsamples are separately used as sources of RNA templates for the reversetranscribing step. In the practice of this method, the results of step(iii) can be normalized to the amount of a constitutively expressed mRNApresent in both said negative control sample and said test sample.Preferred oligonucleotide primers useful for amplifying the TIG3 cDNAhave the sequences of SEQ ID NO:13 and SEQ ID NO:14. In still yetanother preferred embodiment of the invented method the quantitatingstep comprises a nuclease protection assay. More particularly, thenuclease protection assay can include: (a) hybridizing with nucleicacids in the negative control sample and the test sample a singlestranded polynucleotide probe having a sequence complementary to asegment of the sequence of SEQ ID NO:11 linked to a contiguous stretchof nucleotides not complementary to the sequence of SEQ ID NO:11, wherethe complementary sequence is sufficient in length to hybridizespecifically to TIG3 mRNA in an aqueous buffer made 80% formamide; (b)digesting products of hybridizing step (a) with a single-strand-specificnuclease; (c) separating products of digesting step (b) byelectrophoresis; and (d) quantitating the amount of undigested proberemaining after digesting step (b) wherein the undigested probequantitated has a length which corresponds to the sequence of saidsingle-stranded polynucleotide probe which is complementary to thesequence of SEQ ID NO:11. In a particular embodiment, thesingle-strand-specific nuclease is S1 nuclease or RNase. In separatepreferred embodiments of the invented method, the skin cells in theuntreated control and in the culture treated with the test compound canbe psoriatic skin cells, but alternatively may be non-psoriatic skincells. In still yet another preferred embodiment of the invented method,the quantitating step comprises: (i) hybridizing with the negativecontrol sample and the test sample a labeled DNA primer complementary toa segment of a polynucleotide having the sequence of SEQ ID NO:11 toresult in a hybridized primer; (ii) extending the hybridized primer bythe activity of a reverse transcriptase enzyme to produce cDNA; (iii)quantitating the amount of labeled cDNA produced in the extending step,wherein the cDNA quantitated has a length which corresponds to thehumber of nucleotides between the 5′ end of said hybridized primer andthe 5′ terminus of the TIG3 mRNA. In a specific application of thislatter embodiment, the labeled primer can be labeled at its 5′ end.

A third aspect of the invention regards expression vectors forexpressing a TIG3 polypeptide in a eukaryotic cell. These expressionvectors include a polynucleotide which encodes the TIG3 polypeptide,where the TIG3 polypeptide has the amino acid sequence of SEQ ID NO:12,and a promoter operationally linked to this polynucleotide. In oneembodiment, the TIG3 encoding polynucleotide has the sequence of SEQ IDNO:11, and the promoter is a human cytomegalovirus promoter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Overview

Herein we disclose the sequence and utility of a novel polynucleotidethat is induced in a wide variety of cell types by retinoids of the RARsubtype. Expression of this polynucleotide, identified below as“Tazarotene Induced Gene 3” (TIG3), was induced from a low basal levelto a higher level in normal control keratinocytes in vitro and inbiopsies of psoriatic plaques in vivo that had been treated with theanti-psoriatic retinoid called Tazarotene. Significantly, we havediscovered that cell lines derived from human cancers and canceroustumor cells present in several resected tissue samples both expressedthe TIG3 mRNA at reduced levels compared to non-tumor cells. Moreover,cancer-derived cell lines that are growth inhibited by retinoids (i.e.,T47D breast cancer cells) express TIG3 mRNA after retinoid treatment,but cell lines that are not growth inhibited (i.e., MCF7) do not expressTIG3 mRNA in response to retinoid treatment. Importantly, the TIG3polynucleotide and predicted polypeptide sequences were found to berelated to those of a known tumor suppressor gene. These features ofTIG3 gene expression provide a basis for developing nucleic acid andprotein based assays for retinoid activity.

Introduction

As detailed below, we searched for novel retinoid-inducible genesequences in order to obtain reagents useful as indicators of retinoidaction, and to better understand the molecular mechanism of retinoidactivity in skin. This search was directed particularly to the isolationof gene sequences induced in skin culture systems by the anti-psoriaticretinoid called Tazarotene (Esgleyes-Riboty et al., 1994; Weinstein,Brit. J Dermatol. 135(Suppl. 49):32 (1996)). Since Tazarotene was knownto be topically effective for the treatment of psoriasis, we reasonedthat understanding its mechanism of action would provide insight intothe molecular basis of retinoid action in the disease state.

Differential-display PCR (DD-PCR) procedures were used to identifyTazarotene-inducible polynucleotides. As a result of these procedures,we have now identified Tazarotene-induced gene 3 (TIG3), a novel geneproduct that is upregulated by Tazarotene in keratinocyte and skin raftcultures and in cells of biopsied psoriatic skin lesions that had beentreated with Tazarotene. Thus, in addition to inducibility in vitro,TIG3 expression also was induced in vivo by topical treatment ofpsoriatic lesions with an RAR-selective retinoid. Assays for retinoidderivatives that induce the TIG3 mRNA provide a means for identifyingcompounds that can be further investigated as candidates for thetreatment of a wide variety of retinoid responsive diseases.

Analysis of the TIG3 polynucleotide sequence revealed homology to theH-rev107 tumor-suppressor gene. This unexpected relationship initiallysuggested that TIG3 also had tumor suppressor or anti-proliferativeactivity.

Interestingly in view of its structural relatedness to the H-rev107gene, the TIG3 mRNA was expressed in various normal tissues but not incancerous cell lines representing these tissues, or in several primarytumors. Moreover, TIG3 was induced by Tazarotene in breast cancer celllines that responded to the inhibitory effects of retinoids, but not inbreast cancer cell lines that were refractory to retinoid mediatedanti-proliferative activity. Taken together, these results suggestedthat TIG3 may be required for the normal growth control of cells invarious tissues. These results also indicated that assays for detectingTIG3 mRNA inducibility in tumor cells treated with an RAR-selectiveretinoid such as Tazarotene could be adopted for use as a screeningtechnique to identify tumor cells that were responsive to the growthinhibitory effects of retinoids.

One aspect of the present invention regards assays that can identifycompounds having “bioactivity.” As used herein, the term “bioactivity”refers to the ability of a chemical compound to affect an observablechange in a biological system. In this context the synthetic retinoidTazarotene is an example of a drug that exhibits bioactivity in assaysencompassed by the present invention. In particular, Tazarotenestimulates expression of the novel gene, TIG3. Since this activity canbe detected as increased expression of the TIG3 mRNA, Tazarotene is saidto exhibit bioactivity. Drugs that fail to induce the TIG3 mRNA would besaid to exhibit no bioactivity in this assay system.

In addition to nucleic acid-based assays for detecting TIG3 mRNAexpression and induction, we contemplate immunological assays thatemploy anti-TIG3 antibodies as reagents for identifying bioactiveretinoids. In this regard, we contemplate the production of anti-TIG3antibodies for use as reagents in the detection of TIG3 protein. Morespecifically, we contemplate that all or part of the TIG3 cDNA disclosedherein can be operationally ligated to prokaryotic or eukaryoticexpression or gene fusion vectors and introduced into living cells. Theproteins encoded by these vectors can be used as immunogens to elicitthe production of TIG3-specific antibodies. One advantage of usingfusion proteins as immunogens derives from the fact that fusion proteinscan be partly purified more easily than native proteins. Fusion proteinsappropriate for the production of TIG3 immunogens can be any fusionprotein familiar to one having ordinary skill in the art. Such fusionproteins can, for example, comprise glutathione S-transferase (GST)protein sequences as encoded by vectors that are available fromPharmacia (Piscataway, N.J.). Other vectors appropriate for theproduction of TIG3 immunogens can direct the expression of TIG3-proteinA fusion proteins or fusion proteins having metal-binding domains orepitopes recognized by commercially available antibodies.

According to an alternative strategy for the production of anti-TIG3antibodies, synthetic peptides predicted to represent antigenic regionsof the TIG3 protein can be employed as immunogens. For example, thesesynthetic peptides can be coupled to carriers such as keyhole limpethemocyanin (KLH) with MBS (Pierce, Rockford, Ill.) and used asimmunogens for the production of anti-TIG3 antiserum. Antibodiesproduced in this fashion can be used to detect the TIG3 protein by cellstaining, immunoprecipitation and Western blotting protocols. Anti-TIG3antibodies can be used as reagents for detecting the induction of TIG3proteins by retinoids that include Tazarotene, and other compoundshaving potential as therapeutics in the treatment of psoriasis and otherretinoid responsive diseases.

Among the types of diseases contemplated as therapeutic targets ofretinoids that induce TIG3 are: psoriasis, acne, dysplasias and cancers.The category of contemplated dysplasias includes precancerous lesions ofthe epithelial tissues such as oral leukoplakias, dysplasia of thecervix, larynx and bronchi. The category of contemplated cancersincludes carcinomas of the skin, head and neck, cervix, uterus, breastand prostate. Synthetic retinoids may also be therapeutically effectivefor the treatment of atopic dermatitis, allergic rhinitis and asthma.

Finally, another aspect of the present invention relates to recombinantDNA constructs useful for expressing the TIG3 polypeptide in eukaryoticcells. Those having ordinary skill in the art will appreciate thatrecombinant DNA constructs of this sort generally are termed “expressionvectors.” Expression vectors of the invention are structurally relatedin that a polynucleotide encoding a polypeptide having the sequence ofSEQ ID NO:12 is operationally linked downstream of a promoter element. Apromoter element is a DNA element capable of directing transcription inthe nucleus of a cell. By operationally linked it is meant that apolynucleotide translatable as the polypeptide of SEQ ID NO:12 istranscribed from the promoter of the expression vector. This isdistinguished from a case wherein an antisense transcript is producedthat cannot be translated into a polypeptide having the sequence of SEQID NO:12. An exemplary polynucleotide encoding the TIG3 polypeptide hasthe sequence of SEQ ID NO:11. Exemplary promoters useful in connectionwith the invention include: promoters derived from viruses and promotersderived from eukaryotic genomes. Synthetic promoters also arecontemplated for use in connection with the invention. Promoters fallingunder the category of viral promoters include: retroviral promoters,adenoviral promoters, adeno-associated virus promoters and herpes viruspromoters. A human cytomegalovirus (CMV) promoter is specificallycontemplated for operational linkage to a polynucleotide encoding thepolypeptide of SEQ ID NO:12. In a particular embodiment describedherein, an expression vector comprising the human cytomegaloviruspromoter operationally linked to a polynucleotide having the sequence ofSEQ ID NO:11 was created to illustrate the construction of a TIG3expression vector.

Definitions

As used herein, a “hyperproliferative disorder” is a mild or severepathological condition resulting from excessive cell proliferation. Asillustrative examples, psoriasis is a condition associated withkeratinocyte hyperproliferation while a tumor is a conditioncharacterized by hyperproliferation of a clonal population of cells. Aspecific example of a tumor would be a carcinoma, which is a malignanttumor of the epithelium.

As used herein, the phrase “treating a hyperproliferative disorder” isintended to refer to a therapeutic process for improving the symptomsassociated with a hyperproliferative disorder as would be understood byone having ordinary skill in the art. For example, effectively treatingthe hyperproliferative disorder called psoriasis would result in animprovement of the scaling and inflammation associated with the disease.In general, treating a hyperproliferative disorder in the context of theinvention is intended to refer to reducing the rate of cell division incells that are at the root cause of the disorder.

Although other materials and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.General references for methods that can be used to perform the variousnucleic acid manipulations and procedures described herein can be foundin Molecular Cloning: A Laboratory Manual (Sambrook et al., eds. ColdSpring Harbor Lab Publ. 1989) and Current Protocols in Molecular Biology(Ausubel et al., eds., Greene Publishing Associates andWiley-Interscience 1987). The disclosures contained in these referencesare hereby incorporated by reference. A description of the experimentsand results that led to the creation of the present invention follows.

A variety of skin-derived cells were used in the development andpractice of the present invention. The cells employed in theseprocedures served as sources of RNA used as templates for polymerasechain reaction (PCR) amplification procedures and for the analysis ofretinoid-regulated gene expression using a variety of techniques,including blot hybridization and PCR based detection protocols. Unlessspecified otherwise, all cell and tissue samples used in theexperimental procedures described herein were obtained from commercialsources. Primary cultures of human foreskin keratinocytes andfibroblasts were purchased from Clonetics (San Diego, Calif.). Normalskin raft cultures (model ZK 1300), which are three-dimensional humanskin-like tissues, were purchased from Advanced Tissue Sciences (LaJolla, Calif.). While commercial sources of cells and tissues arepreferred for reasons of convenience, primary cultures of human cellsprepared according to the method of the following Example can be usedwith equally good results.

Example 1 describes the methods used to obtain non-transforrned humankeratinocytes and fibroblasts.

EXAMPLE 1 Establishment of Primary Cell Cultures of Human Keratinocytesand Fibroblasts

Fresh human foreskins were washed in ethanol (70%) for 10 secondsfollowed by two washings in keratinocyte growth medium (KGM) purchasedfrom Clonetics (San Diego, Calif.). Tissue samples were cut into smallpieces (4 mm diameter) and incubated with trypsin (0.05% GIBCO-BRL,Grand Island, N.Y.) for 24 hours at 4° C. After digestion, the epidermisthat contained keratinocytes was removed and dissociated into a cellsuspension using sterile forceps. Keratinocytes were pelleted at 1000×gin a Beckman model TJ-6 centrifuge for 6 minutes at 40° C. The resultingcell pellet was then resuspended in KGM and filtered through a 100micron nylon mesh membrane from Tetko, Inc. (New York, N.Y.). Thefiltrate, which contained keratinocytes, was cultured in a T75 tissueculture flask with 20 ml of KGM containing 10% fetal bovine serum (FBS).After three days the culture medium was replaced with KGM that did notcontain FBS. Keratinocytes were maintained in serum-free media and splitat 80% confluence for four passages prior to retinoid treatment.

Fibroblasts were isolated by first cutting the remaining dermis from theforeskins into small pieces using a scalpel, and then incubating withHanks' buffer containing collagenase (0.1% for 1 hour at 37° C.). Thedigest was filtered through a nylon mesh and centrifuged as before.Fibroblasts were resuspended in 20 ml DMEM containing 10% FBS in a T75tissue culture flask. Cells were maintained in DMEM with FBS, split at80% confluence and used for procedures involving treatment withretinoids after 2-3 passages.

In addition to the keratinocyte cultures prepared as described above,three-dimensional reconstructed skin raft cultures were also used inprocedures involving retinoid induction and RNA isolation. Normal skinraft cultures, which are alternatively called “organotypic” or “skinequivalent” cultures, can be obtained commercially. The normal skin raftcultures employed in the procedures described herein were purchased fromAdvanced Tissue Sciences (La Jolla, Calif.).

The structural relationships among the cellular and acellular componentscomprising these skin raft cultures advantageously reproduce some of thecomplex features which characterize skin. In particular, skin raftcultures represent three-dimensional biological models having dermal,epidermal and corneal layers. In the production of these cultures,neonatal fibroblasts were seeded onto an inert nylon mesh and grown intoa dermal tissue. Keratinocytes seeded atop this dermal layer gave riseto an epidermis.

In addition to normal skin raft cultures, we also employed skin raftcultures prepared from psoriatic fibroblasts and normal humankeratinocytes. These “psoriatic skin raft cultures” were perturbed intheir epidermal morphology when compared to their normal counterparts.Thus, these cultures advantageously retained at least some of thepathologic features which characterized the condition of the fibroblastdonor.

The following Example details the method used to isolate the novelretinoid inducible TIG3 polynucleotide. The DD-PCR technique employed inthis procedure involved separately amplifying subsets of the populationof transcripts expressed by mock-treated and Tazarotene-induced humankeratinocytes. Side-by-side electrophoretic separation and visualizationof the amplified cDNAs facilitated comparison of the polynucleotidesrepresented in the two RNA populations. The presence of an amplificationproduct in the lane representing RNA isolated from the induced cell, andthe absence of a corresponding band in the lane representing RNAisolated from mock-treated cells identified a polynucleotiderepresenting a retinoid-induced transcript. As described below, theinducible transcript identified in the following Example unexpectedlywas found to be structurally similar to a known tumor suppressor gene.

Example 2 describes the methods used to identify and isolate apolynucleotide representing a retinoid-inducible gene product.

EXAMPLE 2 Isolation of a Retinoid Responsive Polvnucleotide

Parallel cultures of primary human keratinocytes prepared according tothe method of Example 1 were mock treated with ethanol vehicle alone orinduced with 1 μM Tazarotene (AGN 190168) for three days. At the end ofthe treatment period total RNA was isolated according to standardlaboratory methods and used as a template for 40 cycles of PCRessentially according to the method of Liang et al., (Science 257:967(1992)). Reagents used to conduct the DD-PCR procedure included aHYROGLIPH kit purchased from Genomyx, Inc., (Foster City, Calif.) andEXPAND polymerase purchased from Boehringer Mannheim (Indianapolis,Ind.). We realized that use of a high cycle number was likely tointroduce at lease some replication errors into the sequence of theamplified polynucleotides, but reasoned that the presence of thesesequence errors would not diminish our ability to identify inducibletranscripts. According to a standard protocol that will be familiar tothose having ordinary skill in the art, paired combinations of pooledoligonucleotides were used as primers to amplify cDNAs subsequent to areverse transcription step. Amplification reactions also included[³³P]-dATP so that all reaction products were radiolabeled uniformly.Amplified cDNAs were electrophoretically separated on a long-range 4.5%polyacrylamide gel for 16 hours, and the resulting gel exposed to X-rayfilm to visualize the amplification products.

Autoradiographic results revealed that an amplification product having alength of approximately 0.6 kb was detected only in one of the gel lanesthat had been loaded with products of a reaction primed with RNAisolated from Tazarotene-induced keratinocytes. No equivalent band wasdetected in the lane representing products of a reaction primed with RNAisolated from mock-treated cells. This difference indicated that the 0.6kb amplification product represented an mRNA transcript that was inducedin keratinocytes by Tazarotene, and that had a length of at least 0.6kb.

Inducibility of the mRNA corresponding to the 0.6 kb amplificationproduct was confirmed by Northern analysis of RNA isolated frommock-treated and Tazarotene-induced keratinocytes. The 0.6 kb cDNAfragment was excised from the gel and re-amplified in a PCR procedureusing the gel-isolated cDNA fragment as a template, the combination ofPCR primers that had been employed to amplify the 0.6 kb polynucleotideoriginally, and EXPAND polymerase (Boehringer Mannheim; Indianapolis,Ind.) in a standard PCR amplification procedure. The resultingamplification product was labeled with [³²P]-dCTP (Amersham; ArlingtonHeights, Ill.) by nick translation and then used to probe a Northernblot having one lane each of total RNA isolated from mock-treated andTazarotene-induced keratinocytes, with 15 μg of RNA in each lane. Eventhough the amplification product may have included replication errorssustained as the result of a high number of PCR cycles, these few errorswere not likely to compromise the utility of the amplification productas a hybridization probe. Following hybridization with the labeled probeat 60° C for 2 hours in QUICKHYB hybridization solution (Stratagene; LaJolla, Calif.), the Northern membrane was washed under high stringencyconditions of 0.1×SSC and 1% SDS at 65° C. for 15 minutes and thenexposed to X-ray film.

Northern blotting results indicated that the labeled 0.6 kb probehybridized specifically to a single 0.8-0.9 kb transcript present in theRNA population isolated from Tazarotene induced keratinocytes. A weaklydetectable signal was observed in the lane representing mock-treatedkeratinocyte RNA while a stronger signal was observed in the lanerepresenting Tazarotene-induced RNA. Quantitation of the bandintensities on the X-ray film indicated that the mRNA detected by the0.6 kb probe was increased by at least 4 fold in human keratinocytesfollowing induction with Tazarotene. This result proved that aradiolabeled form of the 0.6 kb amplification product described abovewas useful for specifically detecting a retinoid-inducible mRNA.Specificity of the hybridization was demonstrated by virtue of theunique interaction between the 0.6 kb probe and the single species ofpolynucleotide immobilized on the Northern blot and visualized as asingle band on the autoradiograph. In view of the utility of theamplified polynucleotide as a reagent useful for detectingretinoid-induced gene expression, the amplified polynucleotide wasligated into a plasmid vector and cloned as a step toward furthercharacterizing the structure and function of the corresponding geneproduct.

Notably, the fact that a unique Tazarotene induced mRNA species wasdetected in the Northern blotting procedure described above confirmedthat other hybridization protocols, for example based on dot or slotblotting techniques, would also be useful for detecting expression ofthe TIG3 mRNA.

The 0.6 kb amplification product representing a portion of a 0.8-0.9 kbTazarotene-inducible transcript was ligated into a plasmid vector andcloned according to standard laboratory procedures. More specifically,an aliquot of the amplification reaction that contained the 0.6 kbfragment was combined with the linear form of the pCRII TA CLONINGvector that had been purchased from Invitrogen (San Diego, Calif.),ligated, transformed into competent E. coli host cells and selected onplates containing ampicillin. Several well isolated colonies picked fromone plate represented clones harboring copies of the 0.6 kb fragment asinserts. One of the clones was called pTA-TIG3.

Both strands of the inserts contained in six independent plasmidisolates, pTA-TIG3 being among these isolates, were sequenced instandard dideoxy chain termination protocols using SP6 and T7 sequencingprimers. The polynucleotide sequence of the cDNA insert contained inplasmid pTA-TIG3 is presented as SEQ ID NO:1. A consensus polynucleotidesequence of the 0.6 kb fragment was deduced from alignment andcomparison of the sequences of the six different clonal isolates and ispresented as SEQ ID NO:2. Analysis of this consensus sequence revealedthe presence of a poly(A) addition signal sequence (AATAAA) located 23nucleotides upstream of a poly(A) tail at the 3′ end of thepolynucleotide molecule. In aggregate, these results indicated that thecloned 0.6 kb amplification product represented a partial cDNA that wasmissing polynucleotide sequence information at the 5′ end of themolecule, but was otherwise complete at its 3′ end. The predictedpolypeptide sequence encoded by the polynucleotide of SEQ ID NO:2 ispresented as SEQ ID NO:3.

A preliminary homology search of nucleic acid databases indicated thatthe polynucleotide sequence of the cloned 0.6 kb cDNA fragment wasrelated to the sequence encoding the H-rev107 tumor-suppressor gene.Since the newly cloned polynucleotide may have encoded a protein havingtumor suppressor activity, and in view of the fact that thepolynucleotide was missing sequence information at its 5′ end, wecarried out procedures to isolate a polynucleotide representing themissing upstream sequence that was required to reconstruct the sequenceof the full length cDNA.

A standard PCR protocol for the amplification of 5′ ends of cDNA wasused to obtain polynucleotide sequence information present in the TIG3mRNA, but absent from the partial cDNA clone having the polynucleotidesequence identified herein as SEQ ID NO:2. A first primer having thesequence 5′-TTCACCTCTGCACTGTTGCTC-3′ (SEQ ID NO:4), and corresponding toa region within the coding sequence of the cloned DNA insert of plasmidpTA-TIG3, and an SP6 primer were used to specifically PCR amplify the 5′end of TIG3 from a cDNA library containing total cDNA prepared fromTazarotene-treated skin raft cultures. The cDNA library used for thisprocedure had been constructed in the plasmid vector, pSPORT2(Gibco-BRL, Gaithersburg, Md.). The SP6 primer used for this procedurehad the polynucleotide sequence 5′-ATTTAGGTGACACTATAGAAGAGC-3′ (SEQ IDNO:5). A polynucleotide of approximately 0.3 kb in length was amplified,cloned and sequenced, also according to standard procedures. The 5′ TIG3polynucleotide sequence of this amplification product, presented as SEQID NO:6, contained an ATG translation initiation codon and untranslatedsequence upstream of the coding region. Alignment and combination of thepolynucleotide sequences of SEQ ID NO:2 and SEQ ID NO:6 resulted in apolynucleotide sequence representing the full length TIG3 cDNA,presented here as SEQ ID NO:7. The full length TIG3 polypeptide sequenceis presented as SEQ ID NO:8.

Example 3 describes the method used to obtain a single polynucleotidecorresponding to the cDNA fragments represented by the 0.6 kb cDNAfragment and the 5′ PCR product, and that contained a complete openreading frame. The oligonucleotide primers used in this procedure werederived from the upstream sequence of the 5′ PCR amplification productand the downstream sequence of the 0.6 kb amplification product thatincluded a region representing the 3′ end of the transcript.

EXAMPLE 3 Isolation of a Polynucleotide Containing a Complete OpenReading Frame

First strand cDNA was synthesized using total RNA isolated fromTazarotene-induced keratinocytes as a template and oligo-d(T)₁₆ as aprimer in a standard reverse transcription reaction. Reagents used forsynthesizing cDNA were purchased as a kit from Perkin-Elmer (Norwalk,Conn.). This first strand cDNA then served as a template for a PCRamplification using Pfu DNA polymerase (Stratagene; La Jolla, Calif.)and primers having the sequences of 5′-TTGGATCCTGTGGCTGCTTCAGGCTGTTGC-3′(SEQ ID NO:9) and 5′-TCAAGCTTCCACCATGGCTTCGCCACACCAAGAGCCCA-3′ (SEQ IDNO:10). This second primer contained a Hind III restriction cleavagesite (AAGCTT) and a Kozak consensus sequence (CCACC) to ensure efficienttranslation of an mRNA transcript generated from the amplificationproduct. Those having ordinary skill in the art will appreciate that thePfu DNA polymerase employed in our procedures is a thermostablepolymerase having proofreading activity. Pfu is a high fidelitypolymerase with an error rate substantially lower than the error rate ofTaq polymerase. The amplification product resulting from our procedureshad a length of approximately 0.8 kb as judged by agarose gelelectrophoresis and ethidium bromide staining, and contained the TIG3polynucleotide sequence presented as SEQ ID NO:11. This amplificationproduct was cleaved at the primer-derived restriction endonucleasecleavage sites using Hind III and Bam HI, and samples of the digestedDNA ligated into the pcDNA3 plasmid expression vector (Invitrogen; SanDiego, Calif.) that had been linearized using Hind III and Bam HIrestriction endonucleases. The cDNA insert orientation was such thattranscripts expressed from the plasmid-borne eukaryotic promoter wouldbe translated into a polypeptide having the sequence of SEQ ID NO:12.The pcDNA3-based expression plasmid containing the cDNA insert wascalled pc-TIG3.

Example 4 describes the nucleic acid and protein sequence analysis usedto assess novelty and possible function of the Tazarotene inducible geneproduct isolated according to the methods described above.

EXAMPLE 4 Analysis of the Predicted Protein Encoded by the TIG3Polynucleotide Sequence

The 736 bp long TIG3 cDNA contained an open reading frame extending fromnucleotides 30-521 and encoded a putative protein of 164 amino acids inlength. Computer-assisted homology searches of nucleic acid (Genbank andEMBL) and protein (Swiss-Prot) databases failed to identify any knownDNA or protein sequence that was identical to the TIG3 nucleic acid orpredicted protein sequence. However, this analysis revealed significanthomology between the TIG3 clone and a rat and a human cDNA and protein(RNHrev107 and H-rev107, respectively). The predicted protein encoded bythe TIG3 polynucleotide contained a potential hydrophobic domain betweenamino acids 133-151, a putative cAMP phosphorylation site between aminoacids 160-163, a protein kinase-C (PKC) phosphorylation site betweenamino acids 95-97, and a casein kinase II (CK2) phosphorylation sitebetween amino acids 95-98. The hydropathic profile of the TIG3polypeptide resembled the hydropathic profile of H-rev107. Conservationof the large hydrophobic region in the hydropathic profiles of the TIG3and H-rev107 polypeptides, together with the fact that H-rev107 is knownto be a membrane-associated protein, strongly suggested that TIG3 wasalso a membrane-associated or transmembrane protein. Given theseinteresting structural features of the TIG3 cDNA clone, we proceeded tofurther investigate the nature of retinoid inducibility of the TIG3mRNA.

Example 5 describes the methods used to demonstrate that the TIG3 mRNAwas induced in skin raft cultures selectively by RAR-specific retinoids.

EXAMPLE 5 Induction of TIG3 mRNA by RAR-Specific Retinoids

After demonstrating that TIG3 was induced by Tazarotene in primarykeratinocyte monolayer cultures, we next investigated retinoid inducibleTIG3 mRNA expression in other skin-derived cell culture systems. Moreparticularly, the specificity of the retinoid receptor(s) participatingin the signal transduction pathway leading to TIG3 mRNA induction wasdetermined using a human normal skin raft culture system. RNA samplesisolated from normal skin rafts that had been treated with vehicle aloneor stimulated with a variety of retinoid agonists were assayed for TIG3mRNA induction according to the Northern blotting method describedabove. More specifically, human normal skin rafts were treated for 5days with either the RAR agonist Tazarotene, or RXR agonists AGN 193127or AGN 193193. Retinoids were used at final concentrations of 1 μM.Total RNA isolated from the treated skin rafts was Northern blotted,probed with [³²P]-labeled PCR amplification product representing theTIG3 coding region (SEQ ID NO:11), washed under high stringencyconditions of 0.1×SSC and 1% SDS at 65° C. for 15 minutes and thenexposed to X-ray film to detect TIG3 mRNA transcripts.

Northern blotting results indicated that only the RAR agonist inducedTIG3 mRNA expression. More specifically, we observed that TIG3 mRNA wasinduced at least 4 fold by treatment of normal skin raft cultures withTazarotene. Expression was undetectable by Northern analysis in the RNAsamples isolated from skin raft cultures that had been treated withvehicle alone or with one of the two RXR specific compounds. Thesefindings provided strong evidence that TIG3 mRNA induction was mediatedthrough an RAR-specific signal transduction pathway.

In a related procedure, total RNA that had been isolated from psoriaticskin raft cultures treated with vehicle, Tazarotene, or the free acid ofTazarotene at a concentration of 1 μM for 5 days, was Northern blottedand probed with a labeled TIG3 polynucleotide probe corresponding to the0.6 kb insert in the pTA-TIG3 plasmid. Expression of the TIG3 mRNA wasdetected in the Tazarotene treated lane of the Northern blot, withhigher expression being detected in the sample isolated from the skinraft culture that had been treated with the free acid of Tazarotene.Quantitative analysis of the autoradiograph of the Northern blotindicated that the TIG3 mRNA was induced at least 4 fold in psoriaticskin raft cultures when assayed by Northern blotting using a TIG3polynucleotide probe and high stringency wash conditions of 0.1×SSC and1% SDS at 65° C. for 15 minutes.

A PCR-based assay was used to detect TIG3 mRNA induction as analternative approach for detecting retinoid activity. The extraordinarysensitivity of the PCR technique results from the fact that the quantityof a polynucleotide sequence can be amplified more than a million foldby repetitive cycles of DNA synthesis. By selecting a set of primersthat can be used to amplify a second polynucleotide sequence thatcorresponds to a constitutively expressed mRNA, results effectively canbe normalized between two different reactions. This normalization allowsfor a relative quantitation of the amount of starting material presentin different samples. In the procedure described below, twooligonucleotide primers that could specifically amplify the TIG3polynucleotide present in pooled RNA from patient biopsies weresynthesized for use in RT-PCR protocols. Other primer sets based on theTIG3 cDNA sequence and selected using standard criteria readilyappreciated by those of ordinary skill in the art can also be used todetect TIG3 mRNA expression. Indeed, any oligonucleotide primer set thatcan be used to uniquely amplify a segment of the TIG3 cDNA in a PCRprocedure are anticipated for use in assays for detecting TIG3 mRNAinduction, for example by retinoids. Additionally, the assay can bemodified by substituting skin-derived fibroblasts or normal or psoriaticskin raft cultures or keratinocytes for the biopsy samples describedbelow.

Example 6 describes the method that was used to detect TIG3 mRNAinduction in biopsy samples from patients treated by topical applicationof Tazarotene.

EXAMPLE 6 In Vivo Induction of TIG3 mRNA in Psoriatic Lesions byTazarotene

Retinoid inducibility of the TIG3 mRNA in vivo was verified using a PCRbased assay. A pair of oligonucleotides having sequences5′-GCGACAGCCTGAAGCAGC-3′ (SEQ ID NO:13) and 5′-TTATTGATCCTTCAGTCTTG-3′(SEQ ID NO:14) were prepared for use as primers to amplify a portion ofthe 3′ end of the TIG3 polynucleotide. Oligonucleotide primers foramplifying glyceraldehyde phosphate dehydrogenase (GAPDH) transcripts asa normalization control were purchased from Stratagene, Inc., (La Jolla,Calif.) and used according to manufacturer's instructions.

Patients (n=20) having long-standing bilateral plaque psoriasis weretreated twice daily with vehicle or 0.1% Tazarotene gel in a clinicalstudy for up to 8 weeks. Punch biopsies were taken from 18 patientsafter 2 weeks of treatment and patients were assessed for their clinicalresponse to the drug after 8 weeks. Total pooled RNA from 15 responders(patients with ≧40% decrease in total clinical score at day 56) was usedin standard RT-PCR reactions to amplify the TIG3 and GAPDH transcripts.After oligo-dT primed reverse transcription of the mRNA in the samples,PCR amplification was conducted using either TIG3 or GAPDH primers.Aliquots of the reaction (10 μl) were removed after each cycle beginningat cycle 20. Reaction products were visualized by agarose gelelectrophoresis and ethidium bromide staining.

Results indicated that the TIG3 mRNA was induced in biopsy samples ofTazarotene treated psoriatic plaques. More specifically, a 190 bp TIG3amplification product was detected after only 24 cycles of PCR when RNAisolated from Tazarotene treated plaques served as a template in thereaction. In contrast, the 190 bp amplification product was not detecteduntil 27 cycles in reactions that employed RNA isolated fromvehicle-treated control plaques. This result proved that TIG3 mRNAexpression was induced in psoriatic plaques following treatment withTazarotene. Significantly, the expression of GAPDH in the samples wasunaffected by Tazarotene treatment as judged by detection of the GAPDHamplification product after 24 cycles of PCR in both the control andTazarotene treated biopsies.

Example 7 describes the procedures used to demonstrate that the TIG3mRNA was constitutively expressed in a variety of normal tissues but notin cultured cells derived from malignant tumors.

EXAMPLE 7 TIG3 mRNA is Constitutively Expressed in Normal Tissues butNot in Cell Lines Derived from Human Cancers

Multiple-tissue Northern blots purchased from Clonetech Laboratories,Inc. (Palo Alto, Calif.) were probed with a radiolabeled TIG3polynucleotide probe according to the method of Example 2 to investigateconstitutive expression in various normal tissues. In a separateprocedure, Northern blotted RNA samples isolated from various cell linesderived from human cancers were also probed for TIG3 transcripts. TIG3mRNA expression levels were then compared to expression levels measuredby a similar procedure in non-cancerous tissues. More specifically, RNAsamples used to prepare the Northern blot representing cancer-derivedcell lines were isolated from A549 lung carcinoma cells, SW480colorectal adenoma cells, HL60 cells and K562 cells.

Results indicated that the TIG3 mRNA was easily detected in lanes of theblot representing RNA isolated from normal human lung, liver, kidney,spleen, thymus, prostate, ovary, small intestine, colon and peripheralblood leukocytes. Expression was not detected in heart, brain, placenta,smooth muscle, pancreas or testis. This result indicated that TIG3 mRNAwas constitutively expressed at an easily detectable basal level in atissue-restricted fashion. In contrast to the high levels of TIG3 mRNAexpression detected in normal lung, colon, and peripheral leukocytes,little or no TIG3 mRNA expression was detected in RNA samples isolatedfrom A549 lung carcinoma cells, SW480 colorectal adenoma cells, HL60promyelocytic leukemia or K562 chronic myelocytic leukemia.

Example 8 describes the procedures used to demonstrate that TIG3transcripts were constitutively expressed in non-tumor cells andexpressed at a substantially lower level in tumor cells. As disclosedbelow, a variety of non-tumor and cancerous tissue samples isolated fromhumans were analyzed for TIG3 mRNA expression levels to assessdifferences between the level of expression in malignant andnon-malignant regions of a single surgically resected sample.

EXAMPLE 8 Constitutive Expression of TIG3 mRNA Distinguishes NormalTissue and Primary Tumor Tissue

Total RNA blots containing RNA isolated from resected tumors andadjacent healthy, non-tumor tissue were purchased from Invitrogen (SanDiego, Calif.). The blots were probed according to standard proceduresdescribed above using radiolabeled TIG3 and GAPDH probes. The probe usedfor detecting TIG3 mRNA in this procedure corresponded to the 0.6 kbinsert of plasmid pTA-TIG3. The probe was prepared using a standard PCRamplification protocol in the presence of radiolabeled nucleotides.Tumor samples included: brain, kidney, liver, lung, esophagus, stomach,colon, rectum, bladder, breast, uterus, fallopian, ovarian, thyroid,adrenal, parotid and lymphoma.

Results indicated that TIG3 was constitutively expressed in many normaltissues. More specifically, the TIG3 mRNA was constitutively expressedin kidney, ureter, rectum, uterus, and lymph node. However, TIG3 mRNAexpression was either reduced or undetectable in the cancerouscounterparts of these tissues. Some of the tissues (colon, stomach,breast, liver, fallopian tube, ovary, thyroid and parotid) didconstitutively express TIG3 but did not show any significant differencesin the expression of TIG3 when compared to the cancerous region of thetissue sample.

The decreased expression of TIG3 mRNA in some primary tumors stronglysuggested an anti-proliferative or growth regulatory role for TIG3.Further, it should be noted that TIG3 expression may not be aberrant inall tumors, a situation analogous to other tumor suppressor genes suchas p53 and BRCA1, which are mutated in some, but not all tumors.

Additionally, while TIG3 mRNA was expressed in primary keratinocytesthat served as a normal control, neither constitutive basal levelexpression nor retinoid inducible expression could be detected in HaCaTcells that represented transformed keratinocytes. This finding supportedthe conclusion that the TIG3 gene product possessed antiproliferativeactivity.

In order to further explore the pattern of basal expression and retinoidinducibility of the TIG3 mRNA in the context of hyperproliferativedisorders, we turned to an in vitro system of breast cancer cell lines.Those having ordinary skill in the art will appreciate that someretinoids are known to inhibit the proliferation of a subset of breastcancer cell lines in vitro (Rubin et al. Cancer Res. 54:6549 (1994)). Wereasoned that if the TIG3 polynucleotide encoded a protein havinganti-proliferative activity, then the TIG3 mRNA might be induced byretinoids in cells that were growth-inhibited by retinoids but not incells that were resistant to the growth-inhibitory effects of retinoids.T47D and ZR75-1 are examples of retinoid-sensitive breast cancer celllines that are proliferation-inhibited by Tazarotene or theRAR-selective retinoid called TTNPB (AGN 191183) in a dose-dependentmanner. MCF-7 and MDA-MB-231 are examples of breast cancer cell linesthat are resistant to the anti-proliferative activities of Tazaroteneand TTNPB.

Example 9 describes the method used to demonstrate that the TIG3 mRNAwas selectively induced only in the cancer-derived cell lines that weregrowth-inhibited by exposure to RAR-selective retinoids.

EXAMPLE 9 Tazarotene Induces TIG3 mRNA Expression in Retinoid-ResponsiveBreast Cancer Cells but not in Retinoid Non-Responsive Breast CancerCells

The T47D, ZR75-1, MCF-7 and MDA-MB-231 cell lines were propagated inculture according to standard procedures familiar to those havingordinary skill in the art. Samples of each culture were split two ways,with one aliquot from each culture being treated with vehicle alone as acontrol. The second aliquot from each culture was treated withTazarotene at a concentration of 0.1 μM for a period of three days.Total RNA isolated from each of the vehicle control andTazarotene-treated cultures was Northern blotted and probed withradiolabeled TIG3 and GAPDH probes according to the protocols describedabove. More particularly, the probe used for detecting TIG3 mRNA in thisprocedure corresponded to the 0.6 kb insert of plasmid pTA-TIG3. Theprobe was prepared using a standard PCR amplification protocol in thepresence of radiolabeled nucleotides. Northern blotting resultsindicated that Tazarotene-induced TIG3 mRNA expression was detected onlyin T47D and ZR75-1 cells, but not in MCF-7 or MDA-MB-231 cells.

The experimental results presented to this point indicated that assaysfor TIG3 mRNA induction would be useful for identifying candidate agentsfor treating hyperproliferative disorders. Evidence supporting thisconclusion included the structural similarity between the TIG3 andH-rev107 polynucleotides, the fact that the H-rev107 polynucleotideencoded a protein having antiproliferative activity, and the fact thatthe anti-psoriatic retinoid Tazarotene induced TIG3 mRNA expression inpsoriatic plaques that were characterized by keratinocytehyperproliferation. Accordingly, it would be useful to exploit TIG3 mRNAinducibility in drug screening assays.

The following Example illustrates how blot hybridization protocols canbe used to identify compounds having bioactivity similar to Tazarotene.In this procedure, Tazarotene was used as a positive control forcompounds that induce TIG3 expression. Mock-induction of the samecultured cell type was used as the negative control in this procedure.Test compounds identified as inducers of TIG3 mRNA according to thefollowing procedure are regarded as candidates for the treatment ofhyperproliferative disorders.

In the practice of the method described below, determination of retinoidbioactivity is based on the ability of test compounds to induce TIG3mRNA expression above the level of basal expression detected in RNAsamples isolated from untreated control cells. Accordingly, if an RNAsample isolated from cells treated with a test compound does not containproportionately more TIG3 mRNA than a sample isolated from untreatedcontrol cells, then that test compound will be judged to have nobioactivity. Thus, RNA samples isolated from untreated control cells orcells treated with a compound that does not induce TIG3 mRNA expressionserves as a baseline for measuring inducible expression. Compoundshaving bioactivity similar to that of Tazarotene are identified bycomparing expression of TIG3 mRNA in cells treated with test compoundsand cells treated with Tazarotene as a positive control for TIG3 mRNAinduction.

As described below, the invented assay has been used to prove that atleast one RAR-specific retinoid, in addition to Tazarotene, induced TIG3mRNA expression in psoriatic skin raft cultures. Accordingly, thisRAR-specific retinoid is a candidate agent for treatinghyperproliferative disorders according to the method of the presentinvention. In contrast, compounds that do not substantially induce TIG3mRNA expression above the basal level observed in untreated cells wouldnot be considered for further evaluation as an agent for treatinghyperproliferative disorders according to the invented method.

Example 10 describes a blot hybridization assay for identifyingcompounds that induce TIG3 mRNA expression in psoriatic skin raftcultures. Skin-derived keratinocytes, fibroblasts and normal skin raftcultures can be substituted for the psoriatic skin raft cultures of thefollowing procedure with equally good results.

EXAMPLE 10 Hybridization Assay for Induction of the TIG3 mRNA byRetinoids

Psoriatic skin raft cultures were propagated under standard conditions.Parallel cultures were mock-treated or induced with 1 μM concentrationsof the retinoid to be tested for bioactivity. A mock-treated culture wasused as a negative control to establish basal level expression of TIG3mRNA. The RAR-specific ligand Tazarotene was used as a positive controlfor TIG3 induction. The RAR-specific ligand, AGN 190299 (6-[2-(4,4)dimethyl-thiochroman-6-yl] ethynyl-nicotinic acid), was used as a testcompound in this exemplary procedure. All cultures were induced for fourdays after which time total RNA was isolated by standard laboratoryprocedures. RNA (10 μg) from each of the three samples was separated byelectrophoresis on 1% agarose, 1.1 M formaldehyde gels, transferred tonytran membranes and probed with [³²P]-labeled TIG3 cDNA probecorresponding to the 0.6 kb insert of plasmid pTA-TIG3. The probe wasprepared using a standard PCR amplification protocol in the presence ofradiolabeled nucleotides. The same blots were subsequently stripped andre-probed with [³²P]-labeled GAPDH cDNA probes. Blots were hybridized inQUICKHYB (Stratagene; La Jolla, Calif.) according to manufacturer'sinstructions. Following hybridization with either of the labeled probes,the blots were washed under high stringency conditions in 0.1×SSPE/1%SDS or 0.1×SSC/1% SDS with equally good results. Blots were then exposedto X-ray film. Upon developing. the autoradiographs, similar intensitiesof the GAPDH hybridization signals in all lanes confirmed uniform RNAloading. Accordingly, relative intensities of the TIG3 bands served asdirect indicators of retinoid bioactivity.

Results from the TIG3 Northern hybridization confirmed the lowconstitutive expression and strong inducibility of this mRNA inpsoriatic skin raft cultures. RNA isolated from the culture induced withTazarotene gave a strong TIG3 mRNA signal at 0.8-0.9 kb, as expected. Aband of similar size and slightly greater intensity was observed in thelane corresponding to RNA from the AGN 190299 induced culture. Theseresults indicated that AGN 190299 was strongly bioactive in the TIG3induction assay. Thus, AGN 190299 was identified as a candidate fortherapeutic utility in the treatment of hyperproliferative disorders.

Table 1 summarizes the results of the Northern hybridization procedure.These results confirmed that detection of TIG3 mRNA induction provided ameans for identifying a retinoid that affected gene expression in cellsexhibiting features of the hyperproliferative disorder associated withthe psoriatic phenotype. The TIG3 hybridization probe, used under highstringency conditions, served as an instrument for detecting the TIG3mRNA in these procedures.

TABLE 1 Hybridization Assay for Retinoid Bioactivity: TIG3 mRNAInduction in Psoriatic Skin Rafts Untreated Tazarotene AGN 190299 TIG3(−) (++) (++++) mRNA Expression

Assays that detect induced TIG3 mRNA expression as an indicator ofretinoid bioactivity can employ a variety of nucleic acid probes basedon the sequence of the TIG3 cDNA disclosed herein. In particular, anyprobe having a polynucleotide sequence that can specifically hybridizethe sense strand of the TIG3 cDNA under high stringency conditions isanticipated for use in detecting the TIG3 mRNA in solution or blothybridization protocols. Further, any set of oligonucleotide primersthat can be used to amplify TIG3 polynucleotide sequences starting witha TIG3 mRNA template is also anticipated for use in assays for retinoidbioactivity according to the PCR-based method of our invention.

We particularly note that hybridization probes useful for detecting theTIG3 mRNA need not have a polynucleotide sequence identical to, orperfectly complementary with the polynucleotide sequence given by SEQ IDNO:11. However, hybridization probes useful in connection with theinvention must be sufficiently complementary as to be able to hybridizeto a mRNA comprising the polynucleotide sequence of SEQ ID NO:11. Thelack of requirement for perfect sequence match between the hybridizationprobe and the TIG3 polynucleotide was demonstrated above by virtue ofthe established utility of the 0.6 kb amplification product identifiedby SEQ ID NO:1, a polynucleotide which contained a small number oferrors believed to have been introduced during the PCR amplificationprotocol used to obtain the polynucleotide. In this example, thepolynucleotide probe characterized by SEQ ID NO:1 was not identicallymatched with the sequence of the TIG3 polynucleotide identified by SEQID NO:11. Nonetheless, the probe was sufficiently complementary that asingle species of TIG3 mRNA was detected by Northern blotting.Accordingly, allelic variants of the TIG3 polynucleotides disclosedherein are also contemplated for use as hybridization probes useful fordetecting the TIG3 transcript or cDNA reverse transcribed or amplifiedusing the TIG3 transcript as a template. Those having ordinary skill inthe art will appreciate that allelic variants represent sequencevariants of genes that are located at similar positions on chromosomesof different individuals. Allelic variants are related structurally, andit is this structural relatedness that renders the variants useful ashybridization probes.

Two different approaches can be used to determine the hybridizationcharacteristics of nucleic acid probes useful in the practice of thepresent invention. These approaches can be classified as “theoretical”and “empirical.” Descriptions of the theoretical and empiricalapproaches for determining the melting temperatures of nucleic acidprobes can be found in Molecular Cloning: A Laboratory Manual (Sambrooket al., eds. Cold Spring Harbor Lab Publ. 1989) on pages 11.46 and11.55, respectively. These methods can be used to identifyoligonucleotide primers for use in primer extension and PCR protocols,as well as hybridization probes for use in blotting protocols.

We also contemplate that the aforementioned assay for induction of TIG3mRNA by retinoids can be adapted for identifying candidate therapeuticagents useful for treating tumors in an individual. As indicated above,certain retinoids are known to be useful for inhibiting the growth ofpsoriatic plaques (Boehm et al., Exp. Opin. Invest. Drugs 4:593 (1995))and growth of cells in other hyperproliferative disorders, includingsome tumors (Nagpal et al., Current Pharm. Design 2:295 (1996)).Clearly, it would be of benefit to identify retinoids havinganti-proliferative activity against cells of a tumor biopsy in an invitro system in advance of commencing drug therapy to inhibit tumor cellproliferation in vivo. More particularly, it would be advantageous toknow that a particular retinoid inhibited the growth of cells isolatedfrom a tumor biopsy and cultured in vitro. If a retinoid inhibited cellgrowth, then that retinoid would be considered as a candidate foradministration in vivo for the purpose of inhibiting tumor growth andprogression.

Unfortunately, cell proliferation assays do not lend themselves to highthroughput drug screening assays. Cell proliferation assays involvenumerous steps, including: obtaining a sample of cells representing thehyperproliferative condition, transferring the cells to an in vitroculture, treating a test group of the cells with an agent to be testedfor antiproliferative activity, allowing a period of time to pass andassaying the number of living cells at the end of the time period andcomparing the number of living cells in the test culture with the numberof living cells in a control culture that did not receive the testagent. If there are fewer cells in the culture that received the testagent, when compared with the untreated control sample, then the testagent possessed antiproliferative activity. Conversely, if the test andcontrol cultures contain similar numbers of cells at the end of the timeperiod, then the test agent did not possess antiproliferative activity.Those having ordinary skill in the art will readily appreciate thatsuccess of the proliferation assay critically depends on allowing timeto pass between the addition of the test agent and the final readout.Methods that reduce the time required to allow for cell proliferationwould advantageously accelerate the drug screening assay.

Clearly, it would be advantageous to have available a method for rapidlyidentifying retinoid compounds useful for inhibiting the growth of cellscomprising a patient's tumor. In this way it would be possible to moreselectively identify for therapeutic use a retinoid likely to providetherapeutic benefit.

In one embodiment of the invention primary tumor cells are propagated invitro, administered with a test compound and subsequently assayed forinduction of the TIG3 mRNA. Inducibility of the TIG3 mRNA in tumor cellsisolated from a biopsy and propagated in vitro provides a usefulcriterion for identifying retinoids that are candidates for therapeuticuse in vivo. This method takes advantage of the unexpected correlationbetween the inducibility of the TIG3 mRNA following treatmnent withTazarotene and the susceptibility of cells to the growth inhibitoryactivities of the retinoid. In an exemplary procedure described above,Tazarotene inhibited the proliferation of the T47D and ZR75-1 breastcancer cell lines but did not inhibit the proliferation of the MCF-7 andMDA-MB-231 breast cancer cell lines. Significantly, only the T47D andZR75-1 cell lines were sensitive to the growth-inhibitory activity ofretinoids.

Herein we have disclosed the novel TIG3 polynucleotide and uses thereof.The TIG3 mRNA was found to be induced in by RAR-specific retinoids in avariety of skin-derived cells and tissue systems that included primarykeratinocyte cultures and normal and psoriatic skin raft cultures. Wehave also disclosed that the TIG3 mRNA was induced in vivo in biopsysamples of lesional psoriatic skin that had been treated topically withTazarotene.

In spite of the fact that human keratinocytes express both RARs and RXRs(Fisher et al., J Biol Chem. 269:20629 (1994)), the TIG3 mRNA wasinduced only by RAR-specific retinoids. Accordingly, the TIG3polynucleotide was shown to be an RAR-responsive polynucleotide usefulas a marker for the therapeutic efficacy of retinoids in the treatmentof hyperproliferative disorders, including psoriasis. Assays fordetecting induction of either the TIG3 mRNA or the TIG3 polypeptide willbe useful for the systematic identification of candidate compoundsuseful for treating hyperproliferative disorders such as psoriasis andother diseases.

Contemplated assays for the induction of TIG3 expression can detect theTIG3 mRNA by any of a number of procedures familiar to those havingordinary skill in the art. For example, contemplated assays can detectthe TIG3 mRNA by hybridization protocols such as those disclosed byMeinkoth et al., in Analytical Biochemistby 138:267 (1984), thedisclosure of which is hereby incorporated by reference. Thesehybridization protocols include detection of the TIG3 mRNA in apopulation of immobilized cellular RNA, or alternatively detection ofthe TIG3 mRNA in a sandwich hybridization protocol. In the latterprocedure, an unlabeled probe is affixed to a solid support and servesas a capture probe that hybridizes one region of the TIG3 mRNA. Alabeled nucleic acid probe can bind a different region of the TIG3 mRNAin a detection step.

In addition to the hybridization techniques described above, those ofordinary skill in the art will recognize that a large number of othermethods of TIG3 mRNA detection will be useful in assays for identifyingtest compounds for the treatment of hyperproliferative disorders. Forexample, induction of the TIG3 mRNA can be detected and quantitated bytechniques including S1 assays, RNase protection assays and primerextension assays. Those having ordinary skill in the art will beacquainted with the features and utilities of these assays as theyrelate to RNA detection. General descriptions of these techniques can befound in Molecular Cloning: A Laboratory Manual (Sambrook et al., eds.Cold Spring Harbor Lab Publ. 1989) and Current Protocols in MolecularBiology (Ausubel et al. eds., Greene Publishing Associates andWiley-Interscience 1987) on pages 7.58-7.83.

In S1 and RNase protection assays, the TIG3 mRNA is quantitated byhybridizing RNA samples isolated from cells that are uninduced orinduced with a retinoid such as Tazarotene with labeled nucleic acidprobes harboring sequences complementary to the TIG3 mRNA. If TIG3 mRNAis present in the sample, that mRNA will hybridize to the labeled,complementary nucleic acid strand to form a double-stranded molecule inthe region corresponding to the complementary portion of the probe.Those having ordinary skill in the art will appreciate that an exemplaryhybridization solution useful for hybridizing mRNA and a labeled probeis an aqueous buffer made 80% formamide. Regions of the probe that arenot complementary to the TIG3 mRNA will remain single-stranded. If theprobe is a DNA probe, unhybridized probe and single-stranded regions ofhybridized probe can be digested with S1 nuclease. If the probe is anRNA probe, unhybridized probe and single-stranded regions of hybridizedprobe can be digested with RNase. The protected length of the probe,visualized by autoradiography of digest products that have beenelectrophoretically separated, will be diagnostic of the presence ofTIG3 mRNA. When the hybridization procedure is carried out in probeexcess, quantitative results can be obtained to indicate the presence ofTIG3 mRNA in the starting sample.

In contemplated primer extension assays, an end-labeled oligonucleotideprimer which is complementary to a segment of the TIG3 mRNA ishybridized with samples of RNA isolated from cells that are eitheruninduced or induced with a retinoid such as Tazarotene. If the TIG3mRNA is present in the sample, the oligonucleotide primer will hybridizeto the complementary portion of the TIG3 mRNA. The primer lcan then beextended to the 5′ end of the mRNA by the activity of a reversetranscriptase using the TIG3 mRNA as a template for DNA synthesis. Anextension product of the appropriate size, detected on an autoradiographof electrophoretically separated extension products, will indicate thepresence of TIG3 mRNA in the starting population of RNA.

Labels for nucleic acid hybridization probes of the present inventioncan be any label appropriate for DNA or RNA probes. Such nucleic acidlabels can be radioactive or non-radioactive. Non-radioactive labels canbe detected by a visible color change or by the emission of light ofsufficient intensity that photographic or X-ray film can be exposed.

We also contemplate the use of reporter gene assays to identifyretinoids that, like Tazarotene, activate TIG3 gene expression. As usedherein, a “reporter gene” is a gene that encodes a “reporter” molecule.A “reporter” can be any molecule that can be detected in cells carryingthe corresponding “reporter gene,” but not in cells lacking thatreporter gene. Thus, for example, a reporter can be an enzymne, acolored or fluorescent product, or an antigen that can be detected byantibodies. Reporter gene assays are ideally suited to study theactivity of genes that are regulated at the transcriptional level.

The product of a reporter gene is useful in the study of generegulation. The protein encoded by a reporter gene can also be employedas a surrogate for detecting the products of a different gene.Accordingly, reporter genes can advantageously serve as indirectindicators of gene activity when the reporter is more easily assayedthan the product of the gene of interest. In cases where it is desirableto measure the activity of a weakly expressed gene, or the product of agene for which an assay is not available, a molecular genetic constructthat allows the reporter to be expressed in place of the gene ofinterest can facilitate such measurements. A protein product is commonlythe object of the reporter assay.

Useful reporter molecules may function either as enzymes or as ligandsthat can be detected by tagged antibodies or other ligand-bindingmolecules. Specific examples of reporters that are useful in the studyof gene regulation include bacterial genes such as those encodingchloramphenicol acetyltransferase (CAT) and beta-galactosidase (β-gal),and the firefly luciferase gene. The protein products of all three ofthese reporters can easily be detected by means of simple and sensitiveenzymatic assays. In addition to reporters that are detected by virtueof their enzymatic activities, other reporters can be detected byantibody-based assays.

The Example presented below illustrates one approach that can be used toobtain genomic clones by a standard library screening protocol. As willbe recognized by those having ordinary skill in the art, PCR-basedtechniques provide an alternative method of isolating the TIG3 promoter.

Example 11 illustrates one technique that can be used to isolate thetranscription control region of the TIG3 gene.

EXAMPLE 11 Isolation of the TIG3 Promoter

A nucleic acid segment corresponding to the 5′ end of the TIG3 cDNA isfirst identified for use as a nucleic acid probe according to standardcriteria. This polynucleotide is then radiolabeled to high specificactivity and used as a hybridization probe to identify recombinantclones harboring the 5′ region of the TIG3 cDNA. Some of the genomicclones will also harbor the TIG3 promoter. The transcription initiationsite is identified by S1 nuclease mapping or primer extension analysis.The polynucleotide region upstream of the transcription initiation sitewill possess cis-regulatory elements that confer inducibility of TIG3downstream sequences by retinoids such as Tazarotene.

A reporter gene construct is prepared by ligating the TIG3 transcriptioncontrol region upstream of a reporter gene. Plasmid vectors appropriatefor this purpose can be commercially obtained. For example, thepGL-BASIC vector (Promega) is a vector that harbors the fireflyluciferase coding sequence. This vector would be an appropriaterecipient of the TIG3 promoter according to the contemplated method.

With the availability of reporter constructs as described above, itbecomes possible to create assays for compounds that induce the TIG3promoter.

Example 12 illustrates how reporter gene constructs can be used insensitive assays to identify retinoids that activate the TIG3 promoter.

EXAMPLE 12 Use of TIG3 Reporter Gene Constructs in Assays to IdentifyRetinoids that Stimulate TIG3 Transcription

Stable transfectants harboring the TIG3 promoter/luciferase expressionconstruct of Example 11 are propagated under standard conditions. Aculture of the cells is split into four equal parts, and propagated inseparate flasks. The first flask is left as an untreated control. Asecond flask is treated with Tazarotene as a positive control forinduction of the expression construct. The third and fourth flasks aretreated with test retinoids “A” and “B.” At the end of the treatmentperiod, cells from each of the cultures are harvested and used toprepare cytoplasmic extracts. Luciferase assays are performed usingaliquots of each of the four extracts according to standard protocols.The extract from uninduced cells contains a very low level of luciferaseactivity, while the extract prepared from Tazarotene treated cells has avery high level of activity. These results from the control extractsconfirm that Tazarotene induces the TIG3 promoter. The extract of cellsinduced with retinoid “A” has a level activity similar to the uninducedcontrol extract. This result indicates that retinoid “A” does notexhibit bioactivity in the reporter assay. The extract of cells inducedwith retinoid “B” has a level of luciferase activity comparable to theTazarotene treated cell extract. Retinoid “A” exhibits bioactivity inthe reporter assay.

In addition to assays based on detection of the TIG3 mRNA or reportermolecules, other assays based on retinoid-dependent cell survival arealso contemplated. The approach employed in such assays relies onexpression of a selectable marker under transcriptional control of theTIG3 promoter. For example, we contemplate stable cell lines transfectedwith DNA constructs having the bacterial neomycin drug resistance geneoperationally linked to the TIG3 promoter. High level expression of thedrug resistance marker will depend on activation of the TIG3 promoter.In the absence of the inducer but in the presence of the drug G418, nocell growth will occur. Conversely, in the presence of an inducer of theTIG3 promoter, high level expression of the neomycin resistance genewill permit cell survival in the presence of G418. Accordingly,retinoids that activate the TIG3 promoter will be identified by virtueof their ability to promote cell survival under drug selection.

Bioactive compounds found to induce TIG3 gene expression will beidentified as having potential as therapeutic drugs. The identificationof such bioactive compounds can be made according to assays that detectinduced expression of the TIG3 mRNA or a reporter gene undertranscriptional control of the TIG3 promoter. Alternatively, assaysbased on induction of the TIG3 protein or on cell survival assays asdisclosed above can also be used to identify such compounds. Thefollowing Example describes a procedure that can be used to investigatethe therapeutic potential of the bioactive compounds, such as AGN190299.

Example 13 illustrates how bioactive compounds identified according tothe methods described herein will be tested for therapeutic value in thetreatment of psoriasis. A topical administration protocol is describedbelow.

EXAMPLE 13 Assessing the Therapeutic Potential of Compounds thatStimulate TIG3 Expression

A population of adult volunteers having large psoriatic plaques is firstidentified and then randomly divided into two groups. One group is acontrol group to be treated with a placebo while the other (test) groupis to be treated with a composition containing a bioactive retinoid. Thetreatment protocol and assessment of drug efficacy is performedessentially as described by Esgleyes-Ribot et al., in the Journal of theAmerican Academy of Dermatology 30:581 (1994).

A compound that induces TIG3 gene expression is identified according tomethods such as those disclosed above. This compound is then combinedinto a cream comprising a pharmaceutically acceptable carrier and 0.05%of the bioactive retinoid. The cream is formulated using materials andmethods familiar to one having ordinary skill in the art. A placeboconsists of the carrier alone and does not contain any retinoid.

Participants in the experimental protocol are appropriately treatedeither with the placebo or with the cream containing the retinoid to betested for therapeutic potential. The psoriatic plaques of individualsin the control group are treated with the placebo twice daily for aperiod of two weeks. Similarly, the psoriatic plaques of individuals inthe test group are treated with the composition containing bioactiveretinoid twice daily for the same period.

Individuals administered with the bioactive retinoid show clinicalimprovement after the first two weeks of treatment. In this context,clinical improvement is assessed by erythema, induration and scaling.Immunohistochemical analysis of standard markers indicates thatnormalization of skin histology parallels the clinical improvementobserved in lesions treated with the composition containing thebioactive retinoid. Similarly, expression of inflammatory markers isreduced in biopsies obtained from lesions treated with the compositioncontaining the bioactive retinoid. Few if any members of the controlgroup show clinical improvement. These findings indicate the bioactiveretinoid is effective as a psoriasis treatment, and that treatment ofpsoriatic lesions with the placebo alone had no beneficial effect.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 14(2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 588 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: Other (B) LOCATION: 1...588 (D) OTHER INFORMATION:Polynucleotide sequence of pTA-TIG3 insert (xi) SEQUENCE DESCRIPTION:SEQ ID NO:1: TGAGTACCCC GGGGCTGGCT CCTCCGGTGT CTTCTCAGTC CTGAGCAACAGTGCAGAGGT 60 GAAACGGGGG CGCCTGGAAG ATGTGGTGGG AGGCTGTTGC TATCGGGTCAACAACAGCTT 120 GGACCATGAG TACCAACCAC GGCCCGTGGA GGTGATCATC AGTTCCGCGAAGGAGATGGT 180 TGGTCAGAAG ATGAAGTACA GTATTGTGAG CAGGAACTGT GAGCACTTTGTCGCCCAGCT 240 GAGATATGGC AAGTCCCGCT GTAAACAGGT GGAAAAGGCC AAGGTTGAAGTCGGTGTCGC 300 CACGGCGCTT GGAATCCTGG TTGTTGCTGG ATGCTCTTTT GCGATTAGGAGATACCAAAA 360 AAAAGCAACA GCCTGAAGCA GCCACAAAAT CCTGTGTTAG AAGCAGCTGTGGGGGTCCCA 420 GTGGAGATGA GCCTCCCCCA TGCCTCCAGC AGCCTGACCC TCGTGCCCTGTCTCAGGCGT 480 TCTCTAGATC CTTTCCTCTG TTTCCCTCTC TCGCTGGCAA AAGTATGATCTAATTGAAAC 540 AAGACTGAAG GATCAATAAA CAGCCATCTG CCCCTTCAAA AAAAAAAA 588(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 588 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: Coding Sequence (B) LOCATION: 2...373 (D) OTHER INFORMATION:Consensus sequence derived from six clonal isolates (xi) SEQUENCEDESCRIPTION: SEQ ID NO:2: T GAG TAC CCC GGG GCT GGC TCC TCC GGT GTC TTCTCA GTC CTG AGC AAC 49 Glu Tyr Pro Gly Ala Gly Ser Ser Gly Val Phe SerVal Leu Ser Asn 1 5 10 15 AGT GCA GAG GTG AAA CGG GAG CGC CTG GAA GATGTG GTG GGA GGC TGT 97 Ser Ala Glu Val Lys Arg Glu Arg Leu Glu Asp ValVal Gly Gly Cys 20 25 30 TGC TAT CGG GTC AAC AAC AGC TTG GAC CAT GAG TACCAA CCA CGG CCC 145 Cys Tyr Arg Val Asn Asn Ser Leu Asp His Glu Tyr GlnPro Arg Pro 35 40 45 GTG GAG GTG ATC ATC AGT TCT GCG AAG GAG ATG GTT GGTCAG AAG ATG 193 Val Glu Val Ile Ile Ser Ser Ala Lys Glu Met Val Gly GlnLys Met 50 55 60 AAG TAC AGT ATT GTG AGC AGG AAC TGT GAG CAC TTT GTC ACCCAG CTG 241 Lys Tyr Ser Ile Val Ser Arg Asn Cys Glu His Phe Val Thr GlnLeu 65 70 75 80 AGA TAT GGC AAG TCC CGC TGT AAA CAG GTG GAA AAG GCC AAGGTT GAA 289 Arg Tyr Gly Lys Ser Arg Cys Lys Gln Val Glu Lys Ala Lys ValGlu 85 90 95 GTC GGT GTC GCC ACG GCG CTT GGA ATC CTG GTT GTT GCT GGA TGCTCT 337 Val Gly Val Ala Thr Ala Leu Gly Ile Leu Val Val Ala Gly Cys Ser100 105 110 TTT GCG ATT AGG AGA TAC CAA AAA AAA GCA ACA GCC TGAAGCAGCCACAAAA 389 Phe Ala Ile Arg Arg Tyr Gln Lys Lys Ala Thr Ala 115 120TCCTGTGTTA GAAGCAGCTG TGGGGGTCCC AGTGGAGATG AGCCTCCCCC ATGCCTCCAG 449CAGCCTGACC CTCGTGCCCT GTCTCAGGCG TTCTCTAGAT CCTTTCCTCT GTTTCCCTCT 509CTCGCTGGCA AAAGTATGAT CTAATTGAAA CAAGACTGAA GGATCAATAA ACAGCCATCT 569GCCCCTTCAA AAAAAAAAA 588 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 124 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GluTyr Pro Gly Ala Gly Ser Ser Gly Val Phe Ser Val Leu Ser Asn 1 5 10 15Ser Ala Glu Val Lys Arg Glu Arg Leu Glu Asp Val Val Gly Gly Cys 20 25 30Cys Tyr Arg Val Asn Asn Ser Leu Asp His Glu Tyr Gln Pro Arg Pro 35 40 45Val Glu Val Ile Ile Ser Ser Ala Lys Glu Met Val Gly Gln Lys Met 50 55 60Lys Tyr Ser Ile Val Ser Arg Asn Cys Glu His Phe Val Thr Gln Leu 65 70 7580 Arg Tyr Gly Lys Ser Arg Cys Lys Gln Val Glu Lys Ala Lys Val Glu 85 9095 Val Gly Val Ala Thr Ala Leu Gly Ile Leu Val Val Ala Gly Cys Ser 100105 110 Phe Ala Ile Arg Arg Tyr Gln Lys Lys Ala Thr Ala 115 120 (2)INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (xi) SEQUENCEDESCRIPTION: SEQ ID NO:4: TTCACCTCTG CACTGTTGCT C 21 (2) INFORMATION FORSEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATTTAGGTGACACTATAGAA GAGC 24 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 207 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:6: GGAGCACCAG ACCTCCTCTT GGCTTCGAGATGGCTTCGCC ACACCAAGAG CCCAAACCTG 60 GAGACCTGAT TGAGATTTTC CGCCTTGGCTATGAGCACTG GGCCCTGTAT ATAGGAGATG 120 GCTACGTGAT CCATCTGGCT CCTCCAAGTGAGTACCCCGG GGCTGGCTCC TCCAGTGTCT 180 TCTCAGTCCT GAGCAACAGT GCAGAGG 207(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 736 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: Coding Sequence (B) LOCATION: 30...521 (D) OTHER INFORMATION:(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGAGCACCAG ACCTCCTCTT GGCTTCGAGATG GCT TCG CCA CAC CAA GAG CCC 53 Met Ala Ser Pro His Gln Glu Pro 1 5AAA CCT GGA GAC CTG ATT GAG ATT TTC CGC CTT GGC TAT GAG CAC TGG 101 LysPro Gly Asp Leu Ile Glu Ile Phe Arg Leu Gly Tyr Glu His Trp 10 15 20 GCCCTG TAT ATA GGA GAT GGC TAC GTG ATC CAT CTG GCT CCT CCA AGT 149 Ala LeuTyr Ile Gly Asp Gly Tyr Val Ile His Leu Ala Pro Pro Ser 25 30 35 40 GAGTAC CCC GGG GCT GGC TCC TCC GGT GTC TTC TCA GTC CTG AGC AAC 197 Glu TyrPro Gly Ala Gly Ser Ser Gly Val Phe Ser Val Leu Ser Asn 45 50 55 AGT GCAGAG GTG AAA CGG GGG CGC CTG GAA GAT GTG GTG GGA GGC TGT 245 Ser Ala GluVal Lys Arg Gly Arg Leu Glu Asp Val Val Gly Gly Cys 60 65 70 TGC TAT CGGGTC AAC AAC AGC TTG GAC CAT GAG TAC CAA CCA CGG CCC 293 Cys Tyr Arg ValAsn Asn Ser Leu Asp His Glu Tyr Gln Pro Arg Pro 75 80 85 GTG GAG GTG ATCATC AGT TCT GCG AAG GAG ATG GTT GGT CAG AAG ATG 341 Val Glu Val Ile IleSer Ser Ala Lys Glu Met Val Gly Gln Lys Met 90 95 100 AAG TAC AGT ATTGTG AGC AGG AAC TGT GAG CAC TTT GTC ACC CAG CTG 389 Lys Tyr Ser Ile ValSer Arg Asn Cys Glu His Phe Val Thr Gln Leu 105 110 115 120 AGA TAT GGCAAG TCC CGC TGT AAA CAG GTG GAA AAG GCC AAG GTT GAA 437 Arg Tyr Gly LysSer Arg Cys Lys Gln Val Glu Lys Ala Lys Val Glu 125 130 135 GTC GGT GTGGCC ACG GCG CTT GGA ATC CTG GTT GTT GCT GGA TGC TCT 485 Val Gly Val AlaThr Ala Leu Gly Ile Leu Val Val Ala Gly Cys Ser 140 145 150 TTT GCG ATTAGG AGA TAC CAA AAA AAA GCA ACA GCC TGAAGCAGCC ACAAAA 537 Phe Ala IleArg Arg Tyr Gln Lys Lys Ala Thr Ala 155 160 TCCTGTGTTA GAAGCAGCTGTGGGGGTCCC AGTGGAGATG AGCCTCCCCC ATGCCTCCAG 597 CAGCCTGACC CTCGTGCCCTGTCTCAGGCG TTCTCTAGAT CCTTTCCTCT GTTTCCCTCT 657 CTCGCTGGCA AAAGTATGATCTAATTGAAA CAAGACTGAA GGATCAATAA ACAGCCATCT 717 GCCCCTTCAA AAAAAAAAA 736(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 164 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE:internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Ala Ser Pro His GlnGlu Pro Lys Pro Gly Asp Leu Ile Glu Ile 1 5 10 15 Phe Arg Leu Gly TyrGlu His Trp Ala Leu Tyr Ile Gly Asp Gly Tyr 20 25 30 Val Ile His Leu AlaPro Pro Ser Glu Tyr Pro Gly Ala Gly Ser Ser 35 40 45 Gly Val Phe Ser ValLeu Ser Asn Ser Ala Glu Val Lys Arg Gly Arg 50 55 60 Leu Glu Asp Val ValGly Gly Cys Cys Tyr Arg Val Asn Asn Ser Leu 65 70 75 80 Asp His Glu TyrGln Pro Arg Pro Val Glu Val Ile Ile Ser Ser Ala 85 90 95 Lys Glu Met ValGly Gln Lys Met Lys Tyr Ser Ile Val Ser Arg Asn 100 105 110 Cys Glu HisPhe Val Thr Gln Leu Arg Tyr Gly Lys Ser Arg Cys Lys 115 120 125 Gln ValGlu Lys Ala Lys Val Glu Val Gly Val Ala Thr Ala Leu Gly 130 135 140 IleLeu Val Val Ala Gly Cys Ser Phe Ala Ile Arg Arg Tyr Gln Lys 145 150 155160 Lys Ala Thr Ala (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:9: TTGGATCCTG TGGCTGCTTC AGGCTGTTGC 30(2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION:SEQ ID NO:10: TCAAGCTTCC ACCATGGCTT CGCCACACCA AGAGCCCA 38 (2)INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:505 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:Coding Sequence (B) LOCATION: 1...492 (D) OTHER INFORMATION: (xi)SEQUENCE DESCRIPTION: SEQ ID NO:11: ATG GCT TCG CCA CAC CAA GAG CCC AAACCT GGA GAC CTG ATT GAG ATT 48 Met Ala Ser Pro His Gln Glu Pro Lys ProGly Asp Leu Ile Glu Ile 1 5 10 15 TTC CGC CTT GGC TAT GAG CAC TGG GCCCTG TAT ATA GGA GAT GGC TAC 96 Phe Arg Leu Gly Tyr Glu His Trp Ala LeuTyr Ile Gly Asp Gly Tyr 20 25 30 GTG ATC CAT CTG GCT CCT CCA AGT GAG TACCCC GGG GCT GGC TCC TCC 144 Val Ile His Leu Ala Pro Pro Ser Glu Tyr ProGly Ala Gly Ser Ser 35 40 45 AGT GTC TTC TCA GTC CTG AGC AAC AGT GCA GAGGTG AAA CGG GGG CGC 192 Ser Val Phe Ser Val Leu Ser Asn Ser Ala Glu ValLys Arg Gly Arg 50 55 60 CTG GAA GAT GTG GTG GGA GGC TGT TGC TAT CGG GTCAAC AAC AGC TTG 240 Leu Glu Asp Val Val Gly Gly Cys Cys Tyr Arg Val AsnAsn Ser Leu 65 70 75 80 GAC CAT GAG TAC CAA CCA CGG CCC GTG GAG GTG ATCATC AGT TCT GCG 288 Asp His Glu Tyr Gln Pro Arg Pro Val Glu Val Ile IleSer Ser Ala 85 90 95 AAG GAG ATG GTT GGT CAG AAG ATG AAG TAC AGT ATT GTGAGC AGG AAC 336 Lys Glu Met Val Gly Gln Lys Met Lys Tyr Ser Ile Val SerArg Asn 100 105 110 TGT GAG CAC TTT GTC GCC CAG CTG AGA TAT GGC AAG TCCCGC TGT AAA 384 Cys Glu His Phe Val Ala Gln Leu Arg Tyr Gly Lys Ser ArgCys Lys 115 120 125 CAG GTG GAA AAG GCC AAG GTT GAA GTC GGT GTG GCC ACGGCG CTT GGA 432 Gln Val Glu Lys Ala Lys Val Glu Val Gly Val Ala Thr AlaLeu Gly 130 135 140 ATC CTG GTT GTT GCT GGA TGC TCT TTT GCG ATT AGG AGATAC CAA AAA 480 Ile Leu Val Val Ala Gly Cys Ser Phe Ala Ile Arg Arg TyrGln Lys 145 150 155 160 AAA GCA ACA GCC TGAAGCAGCC ACA 505 Lys Ala ThrAla (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 164 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE:internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Ala Ser Pro HisGln Glu Pro Lys Pro Gly Asp Leu Ile Glu Ile 1 5 10 15 Phe Arg Leu GlyTyr Glu His Trp Ala Leu Tyr Ile Gly Asp Gly Tyr 20 25 30 Val Ile His LeuAla Pro Pro Ser Glu Tyr Pro Gly Ala Gly Ser Ser 35 40 45 Ser Val Phe SerVal Leu Ser Asn Ser Ala Glu Val Lys Arg Gly Arg 50 55 60 Leu Glu Asp ValVal Gly Gly Cys Cys Tyr Arg Val Asn Asn Ser Leu 65 70 75 80 Asp His GluTyr Gln Pro Arg Pro Val Glu Val Ile Ile Ser Ser Ala 85 90 95 Lys Glu MetVal Gly Gln Lys Met Lys Tyr Ser Ile Val Ser Arg Asn 100 105 110 Cys GluHis Phe Val Ala Gln Leu Arg Tyr Gly Lys Ser Arg Cys Lys 115 120 125 GlnVal Glu Lys Ala Lys Val Glu Val Gly Val Ala Thr Ala Leu Gly 130 135 140Ile Leu Val Val Ala Gly Cys Ser Phe Ala Ile Arg Arg Tyr Gln Lys 145 150155 160 Lys Ala Thr Ala (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:13: GCGACAGCCT GAAGCAGC 18 (2)INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO:14: TTATTGATCC TTCAGTCTTG 20

What is claimed is:
 1. An isolated polynucleotide encoding a proteinhaving the amino acid sequence SEQ ID NO:12.
 2. The isolatedpolynucleotide of claim 1, comprising a nucleotide base sequence SEQ IDNO:11.
 3. The isolated polynucleotide of claim 1, consisting of anucleotide sequence SEQ ID NO:11.
 4. A recombinant or syntheticpolypeptide selected from the group consisting of: i) a polypeptidecomprising an amino acid sequence consisting of SEQ ID NO:12, ii) afusion polypeptide comprising an epitope able to elicit production of anantibody by an antibody-producing cell wherein said antibody willspecifically bind a polypeptide having an amino acid sequence consistingof SEQ ID NO:12 and said epitope consists of an amino acid sequencecontained within SEQ ID NO:12, and iii) an immunogenic polypeptidecomprising an epitope able to elicit production of an antibody by anantibody-producing cell wherein said antibody will specifically bind apolypeptide having an amino acid sequence consisting of SEO ID NO:12 andsaid epitope consists of an amino acid sequence contained within SEQ IDNO:12.
 5. The polypeptide of claim 4 in which the polypeptide consistsof a fusion protein.
 6. The fusion protein of claim 5 further comprisingat least a ligand-binding portion of another protein selected from thegroup consisting of: i) glutathione S-transferase, ii) protein A, iii)proteins having metal-binding domains, iv) proteins having epitopesrecognized by commercially available antibodies.
 7. The polypeptide ofclaim 4 wherein said polypeptide is a recombinant polypeptide.
 8. Therecombinant polypeptide of claim 7 which comprises an amino acidsequence consisting of: SEQ ID NO:12.
 9. The recombinant polypeptide ofclaim 7 which comprises an immunogenic peptide which is encoded by SEQID NO:11.
 10. The polypeptide of claim 4 wherein said polypeptide is asynthetic polypeptide.
 11. The synthetic polypeptide of claim 10comprising a polypeptide comprising an amino acid sequence consistingof: SEQ ID NO:12.
 12. The synthetic polypeptide of claim 10 whichfurther comprises a carrier coupled therewith.
 13. The syntheticpolypeptide of claim 12 wherein said carrier comprises keyhole limpethemocyanin.